Project description:Mesenchymal stromal cells (MSCs) are located in bone marrow where they help to maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal linages. The signalling mechanisms that enable precise control of MSC function remain unclear. Here, we have identified a non-canonical epidermal growth factor (EGF) signalling pathway in MSCs, which acts via integrin-linked kinase (ILK) to activate β-catenin, a key component of Wnt signalling. EGF induces nuclear translocation of β-catenin in MSCs but does not drive T cell factor (TCF)-mediated transactivation of Wnt target genes, and we demonstrate by Design of Experiments statistical analysis that the EGF/β-catenin and Wnt/β-catenin pathways do not cross-talk following co-stimulation with multiple concentrations of both ligands. By examining EGF-regulated genes in MSCs by RNA-Sequencing, we identified gene sets that were exclusively regulated by the EGF/b-catenin pathway, which were distinct from canonical EGF-regulated genes. In contrast, the expression of subsets of canonical EGF signalling gene targets were significantly influenced by b-catenin activation. These newly-identified EGF signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by canonical EGF signalling. Overall design: Examination of expression profiles of 3 samples of untreated MSCs, 3 samples EGF treated, 3 samples EGF + IWR-1 treated MSCs using RNA-seq analysis
Project description:Integrin-linked kinase (ILK) links integrins to the actin cytoskeleton and is believed to phosphorylate several target proteins. We report that a keratinocyte-restricted deletion of the ILK gene leads to epidermal defects and hair loss. ILK-deficient epidermal keratinocytes exhibited a pronounced integrin-mediated adhesion defect leading to epidermal detachment and blister formation, disruption of the epidermal-dermal basement membrane, and the translocation of proliferating, integrin-expressing keratinocytes to suprabasal epidermal cell layers. The mutant hair follicles were capable of producing hair shaft and inner root sheath cells and contained stem cells and generated proliferating progenitor cells, which were impaired in their downward migration and hence accumulated in the outer root sheath and failed to replenish the hair matrix. In vitro studies with primary ILK-deficient keratinocytes attributed the migration defect to a reduced migration velocity and an impaired stabilization of the leading-edge lamellipodia, which compromised directional and persistent migration. We conclude that ILK plays important roles for epidermis and hair follicle morphogenesis by modulating integrin-mediated adhesion, actin reorganization, and plasma membrane dynamics in keratinocytes.
Project description:Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (<or=1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and beta-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.
Project description:Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function.
Project description:The Wnt/beta catenin pathway has been highlighted as an important player of brain tumors aggressiveness and resistance to therapies. Increasing knowledges of the regulation of beta-catenin transactivation point out its hub position in different pathophysiological outcomes in glioma such as survival and migration. Crosstalks between integrins and beta-catenin pathways have been suggested in several tumor tissues. As we demonstrated earlier that ?5?1 integrin may be considered as a therapeutic target in high grade glioma through its contribution to glioma cell migration and resistance to chemotherapy, we addressed here the potential relationship between ?5?1 integrin and beta-catenin activation in glioma cells. We demonstrated that overexpression and activation by fibronectin of ?5?1 integrin allowed the transactivation of beta-catenin gene targets included in an EMT-like program that induced an increase in cell migration. Hampering of beta catenin activation and cell migration could be similarly achieved by a specific integrin antagonist. In addition we showed that ?5?1 integrin/AKT axis is mainly involved in these processes. However, blockade of beta-catenin by XAV939 (tankyrase inhibitor leading to beta-catenin degradation) did not synergize with p53 activation aiming to cell apoptosis as was the case with integrin antagonists. We therefore propose a dual implication of ?5?1 integrin/AKT axis in glioma cell resistance to therapies and migration each supported by different signaling pathways. Our data thus suggest that ?5?1 integrin may be added to the growing list of beta-catenin modulators and provide new evidences to assign this integrin as a valuable target to fight high grade glioma.
Project description:Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin ?1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.
Project description:The tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) has been defined by its ability to promote tumorigenesis on carcinogen-initiated mouse skin. Activation of Wnt/?-catenin signaling has a decisive role in mouse skin carcinogenesis, but it remains unclear how TPA activates Wnt/?-catenin signaling in mouse skin carcinogenesis. Here, we found that TPA could enhance Wnt/?-catenin signaling in a casein kinase 1 (CK1) ?/?-dependent manner. TPA stabilized CK1? and enhanced its kinase activity. TPA further induced the phosphorylation of LRP6 at Thr1479 and Ser1490 and the formation of a CK1?-LRP6-axin1 complex, leading to an increase in cytosolic ?-catenin. Moreover, TPA increased the association of ?-catenin with TCF4E in a CK1?/?-dependent way, resulting in the activation of Wnt target genes. Consistently, treatment with a selective CK1?/? inhibitor SR3029 suppressed TPA-induced skin tumor formation in vivo, probably through blocking Wnt/?-catenin signaling. Taken together, our study has identified a pathway by which TPA activates Wnt/?-catenin signaling.
Project description:BACKGROUND:Integrin-mediated platelet-tumor cell contacting plays an important role in promoting epithelial-mesenchymal transition (EMT) transformation of tumor cells and cancer metastasis, but whether it occurs in breast cancer cells is not completely clear. OBJECTIVE:The purpose of this study was to investigate the role of integrin ?2?1 in platelet contacting to human breast cancer cell line MCF-7 and its effect on the EMT and the invasion of MCF-7 cells. METHODS:Human platelets were activated by thrombin, and separated into pellets and releasates before the co-incubation with MCF-7 cells. Cell invasion was evaluated by transwell assay. The surface integrins on pellets and MCF-7 cells were inhibited by antibodies. The effect of integrin ?2?1 on Wnt-?-catenin pathway was assessed by integrin ?2?1-silencing and Wnt-?-catenin inhibitor XAV. The therapeutic effect of integrin ?2?1-silencing was confirmed in the xenograft mouse model. RESULTS:Pellets promote the invasion and EMT of MCF-7 cells via direct contacting of surface integrin ?2?1. The integrin ?2?1 contacting activates Wnt-?-catenin pathway and promotes the expression of EMT proteins in MCF-7 cells. The activated Wnt-?-catenin pathway also promotes the autocrine of TGF-?1 in MCF-7 cells. Both Wnt-?-catenin and TGF-?1/pSmad3 pathways promote the expression of EMT proteins. Integrin ?2?1-silencing inhibits breast cancer metastasis in vivo. CONCLUSIONS:The direct interaction between platelets and tumor cells exerts its pro-metastatic function via surface integrin ?2?1 contacting and Wnt-?-catenin activation. Integrin ?2?1-silencing has the potential effect of inhibiting breast cancer metastasis.
Project description:OBJECTIVES:Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has significant therapeutic efficacy in non-small-cell lung cancer (NSCLC) patients. However, acquired resistance is inevitable and limits the long-term efficacy of EGFR-TKI. Our study aimed to investigate the role of ras-associated binding protein 25 (Rab25) in mediating EGFR-TKI resistance in NSCLC. MATERIALS AND METHODS:Rab25 expression in NSCLC patients was measured by immunohistochemical staining. Western blotting was used to analyse the expression of molecules in the Rab25, EGFR and Wnt signalling pathways. Lentiviral vectors were constructed to knock in and knock out Rab25. The biological function of Rab25 was demonstrated by cell-counting kit-8 and flow cytometry. The interaction between Rab25 and ?1 integrin was confirmed by co-immunoprecipitation. RESULTS:Rab25 overexpression induced erlotinib resistance, whereas Rab25 knockdown reversed this refractoriness in vitro and in vivo. Moreover, Rab25 interacts with ?1 integrin and promotes its trafficking to the cytoplasmic membrane. The membrane-?1 integrin induced protein kinase B (AKT) phosphorylation and subsequently activated the Wnt/?-catenin signalling pathway, promoting cell proliferation. Furthermore, high Rab25 expression was associated with poor response to EGFR-TKI treatment in NSCLC patients. CONCLUSIONS:Rab25 mediates erlotinib resistance by activating the ?1 integrin/AKT/?-catenin signalling pathway. Rab25 may be a predictive biomarker and has potential therapeutic value in NSCLC patients with acquired resistance to EGFR-TKI.
Project description:A receptive endometrium is a prerequisite for successful embryo implantation, and about one-third of repeated embryo implantation failure attribute to defective endometrial receptivity. Integrin-linked kinase (ILK), a 59kDa serine/threonine-protein kinase, plays a vital role in multiple cellular processes, including cell proliferation, apoptosis, and invasion. However, its role in endometrial receptivity is still unclear. In the current study, we demonstrated that ILK level was significantly downregulated in the serum of patients with unexplained infertility compared with healthy non-pregnancy. Functionally, ILK knockdown inhibited endometrial epithelial cells (EECs) proliferation and invasion, whereas ILK overexpression promoted endometrial EECs proliferation and invasion. ILK inhibition also repressed the adhesion rate of embryonic cells to EECs. In vivo studies further demonstrated that ILK inhibition suppressed endometrium receptivity formation and embryo implantation potential. Mechanistically, the downregulation of ILK inactivated Wnt/?-catenin signaling and thus resulted in the downregulation of MMP-3 and MMP-9 expression. Importantly, activation of Wnt/?-catenin signaling, partially recovered ILK inhibition-caused endometrium receptivity defects, and embryo implantation failure. Considered all the current data, it verified that the low expression of ILK exacerbates endometrial receptivity formation by inactivating Wnt/?-catenin signaling and decreasing the MMP-3/9 expression and indicated that ILK may be applied as an indicator of endometrial receptivity, and as a diagnostic and therapeutic target for infertility.